At all stages of their development, cells of the nephrone are coated by a basement membrane, an extracellular matrix containing collagen IV alpha chain trimers laminin alpha-beta-gamma chain trimers, entactin/nidogen, and a heparan sulfate proteoglycan. Components of the basement membrane are involved in glomerular development in the embryo and contribute to glomerular function in the adult. Collagen IV alpha chain trimers form a network which is the most abundant component of all basement membranes. These are six genetically distinct alpha chains that have been identified. Two, the alpha1 and alpha2 chains, are found widely in basement membranes throughout the body. The other four, alpha3-alpha6, exhibit a restricted pattern of accumulation, including a subset of kidney basement membranes: alpha3-alpha5 are found in the glomerular basement membrane (GMB) and in a subset of tubules, while alpha6 is absent from the GBM but is present in Bowman's capsular basement membrane and in some tubules. In humans, the hereditary glomerulonephritis known as Alport syndrome is caused my mutation in any one of the collagen alpha3-alpha5 chains, underscoring their importance in GBM function. Several years ago we began an effort to study the structure, regulation, and function of the collagen alpha3- alpha5(IV) chains in mice. This initial effort has culminated in the generation of a knockout mouse with a targeted mutation in the collagen alpha3(IV) gene. These mice exhibit many features of human Alport syndrome, including delayed onset nephritis leading to end stage renal disease, an absence of collagen alpha3-alpha5(IV) immunoreactivity in the kidney and in the lung, and in a split, thickened GBM with a characteristic "basket weave" appearance. We now propose to use these mice to gain new insights into the mechanism of collagen IV chain assembly and into the pathogenesis of Alport syndrome. We will make cell lines from the mutant mice and use them as a model system for determining which portions of collagen alpha3(IV) are necessary for proper assembly of the collagen alpha3-alpha5(IV) network. We will attempt to rescue the mutation in vivo by making transgenic mice that express cDNAs encoding collagen alpha3(IV). Finally, the effect of genetic background on progression to end stage renal disease, which we have identified in preliminary studies, will be characterized in detail. The results obtained under the auspices of this proposal will have important implication for the pathogenesis of Alport syndrome in man and for approaches current under development that will use gene therapy to treat the human disease.